PROJECT SUMMARY/ABSTRACT Human papillomavirus (HPV) is the most common sexually transmitted infection. Persistent HPV infection is prevalent in young men and women and infection with a high-risk HPV type, especially HPV-16, is associated with an increased risk of developing an intraepithelial neoplasia. Furthermore, immunosuppressed individuals have a greater risk of being infected with HPV and subsequently developing HPV-associated precancer and cancer lesions. Currently, there is no antiviral treatment for persistent genital HPV infection. The current management for such patients only includes repeat screening for the development precancer and cancer lesions, which generates significant discomfort and anxiety. Furthermore, 90% of all HPV-associated vaginal, vulval and anal cancers are attributable to HPV-16. Additionally, while surgical treatment is quite effective for precancer and early cancer lesions of the cervix, it is associated with significant morbidity and high recurrence rates in vaginal, vulval and anal intraepithelial lesions. Thus, there is an urgent need to develop an innovative treatment to eliminate persistent, high-risk HPV infections, especially HPV-16, in the vagina, vulva and anus. In order to develop an effective treatment for persistent HPV-16 infection and HPV-16-associated anogenital lesions, it is essential to develop a preclinical model of chronic HPV infection. It is now clear that high-risk HPV E6 and E7 oncogenic proteins are responsible for the malignant progression of HPV-associated lesions. We recently generated an HPV-16 pseudovirion (psV) carrying a DNA construct capable of expressing luciferase, HPV E6 and E7 oncogenes and Ras oncogene individually. We found that mice infected with this HPV-16 psV persistently expressed luciferase as well as E6 and E7 oncogenes 150 days after infection of the vagina. In comparison, HPV-16 psV carrying control DNA with luciferase only demonstrated transient expression of the encoded gene. Thus, we have created a non-invasive imaging system to follow the persistent expression of E6/E7 in the vagina. The purpose of the current project is to characterize whether the animal model resembles persistent anogenital HPV infection and to investigate the molecular pathogenesis and immune responses as persistent infection progresses to precancer and cancer lesions. Our specific aims are to: (1) characterize the molecular pathogenesis of persistent HPV-16 E6/E7 and Ras expression in the vagina, vulva and anus of immunocompetent and immunocompromised hosts and (2) characterize E6 and E7-specific humoral and cell- mediated immune responses following persistent anogenital infection with HPV-16 psVs. The successful implementation of this project will generate a preclinical model of persistent HPV-16 E6/E7 expression, resembling chronic high risk HPV infection. This will represent a truly innovative methodology that may change the paradigm in HPV research. Additionally, our preclinical model will facilitate the development of molecular interventions targeting E6/E7 oncogenic proteins for the control of HPV-associated diseases. The results of this study will have significant implications for the management and treatment of persistent HPV infection.